Method of screening ptp ç activitiy promoter or inhibitor

ABSTRACT

An object of the present invention is to provide a remedy for dysfunction of central monoamine pathway, a method for screening a PTPζ inhibitor or activator, which is useful as a remedy for gastric ulcer caused by  Helicobacter pylori  or pleiotrophin which is a heparin-binding secretory protein, and a non-human model animal being hyposensitive to a stimulant drug, VacA which is a toxin of  Helicobacter pylori , or pleiotrophin by utilizing the physiological function of PTPζ. After administering a subject material to PTPζ knockout mice and wild-type mice, PTPζ activity in the PTPζ knockout mice and the wild-type mice is compared and evaluated to screen a PTPζ inhibitor or activator. Examples of the comparison and the evaluation of the PTPζ activity include the comparison and the evaluation of the function of central monoamine pathway such as changes in the level of central monoamine metabolism, sensitivity to a stimulant drug, the presence of dysfunction of mesolimbic dopamine pathway, level of acclimation to new circumstances, or stress-responsiveness, and the comparison and the evaluation of the level of binding to VacA, a toxin of  Helicobacter pylori,  or pleiotrophin.

TECHNICAL FIELD

[0001] The present invention relates to a remedy for dysfunction ofcentral monoamine pathway with the use of a non-human animal such as amouse or the like which is generated by a homologous recombinationtechnique for genes and is deficient in its receptor-type proteintyrosine phosphatase (PTPζ/RPTPβ) gene, a screening of a remedy forgastric ulcer caused by Helicobacter pylori or the like, a non-humanmodel animal being hyposensitive to a central stimulant drug (anaddictive drug) and a non-human model animal being hyposensitive toVacA, a toxin of Helicobacter pylori, or the like.

BACKGROUND ART

[0002] When a cell receives a stimulus from outside, its intracellularsignaling pathway is activated to induce proliferation, differentiation,apoptosis and the like of the cell. Tyrosine phosphorylation ofintracellular proteins acts an extremely important role in variousphases of the signaling pathway, and the state of tyrosinephosphorylation of each protein is always regulated by dynamicequilibrium of delicate balance of two families of enzymes, tyrosinekinase (PTK) and tyrosine phosphatase (PTP). It is known that thistyrosine phosphorylation of proteins is involved in controlling theefficiency of neural circuit formation and neurotransmission in brains(SEITAI NO KAGAKU Vol. 48, No. 6, 534-538, (1997); PROTEIN, NUCLEIC ACIDAND ENZYME Vol. 43, No. 8, 1136-1143 (1998)), and is important for theformation and the maintenance of the functions in an immune system andother organs (PROTEIN, NUCLEIC ACID AND ENZYME Vol. 43, No. 8, 1131-1135(1998)). On the other hand, it is reported that the abnormal tyrosinephosphorylation of proteins is involved in defects in neural circuitformation, disturbance of memory and learning, abnormal apoptosis,tumorigenesis or the like (PROTEIN, NUCLEIC ACID AND ENZYME Vol. 43, No.8, 1186-1192 (1998)).

[0003] To date, more than 80 kinds of PTP have been identified, and itis presumed that the number of genes of PTP in human would reach to 500.Similar to PTK, PTP is classified into two types: a receptor type and anon-receptor type. A receptor-type PTP has two or one enzymic domainintracellularly, and is classified into several groups according to thecharacteristics of its extracellular domain. PTPζ, which has a carbonicanhydrase domain in N-terminal, has been identified as a receptor-typetyrosine phosphatase specific to the central nervous system. Theinventors of the present invention have reported that PTPζ is a receptorof growth factors including pleiotrophin and midkine (J. Biol. Chem.271, 21446-21452, 1996; J. Cell Biol. 142, 203-216, 1998; J. Biol. Chem.274, 12471-12479, 1999). In addition, PTPζ is known to interact withcell adhesion molecules which belong to the immunogloblin super family,such as N-CAM, and is thought to be responsible for important functionsin differentiation, migration and neurotransmission of neurons. Thepresent inventors have already generated a PTPζ gene-deficient mouse andreported that PTPζ has expressed in both neurons and astrocytes(Neuroscience Letters 274, 135-138, 1998). The PTPζ gene-deficient mousehas grown and propagated normally, and no major morphologic abnormalityhas been identified. However, the physiological role of PTPζ has beenhardly elucidated so far.

[0004] Recently, Hirayama has shown a possibility that PTPζ functions asa receptor of VacA, an exotoxin secreted by Helicobacter pylori which iswell known as a cause of gastric ulcer, through a test system using celllines (J. Biol. Chem. 274, 36693-36699, 1999). VacA, a toxin ofHelicobacter pylori, is detected in about 90% of patients who sufferfrom acute gastritis or gastric ulcer, and it is reported that a mouseorally administered with VacA, a toxin extracellulary secreted byHelicobacter pylori, shows the onset of acute gastritis.

[0005] The clarification of in vivo role of PTPζ, whose physiologicalfunction has been conventionally unknown, makes it possible to providefindings and experimental materials that lead to the elucidation ofonset mechanisms of diseases related to the physiological function ofPTPζ, and to the development of remedies of the diseases. An object ofthe present invention is to provide a remedy for dysfunction of centralmonoamine pathway, a method for screening a PTPζ inhibitor or activator,which is useful as a remedy for gastric ulcer caused by Helicobacterpylori or the like, as a non-human model animal being hyposensitive to acentral stimulant drug (an addictive drug) and as a non-human modelanimal being hyposensitive to VacA, a toxin of Helicobacter pylori, orthe like, by utilizing the physiological function of PTPζ identifiedwith the use of a PTPζ gene-deficient mouse.

[0006] As aforementioned, the present inventors have already generatedthe mouse with the use of a homologous recombination technique, but itwas difficult to eliminate the influence of other genes from the mousebecause the mouse was a hybrid wherein chromosomes of two lines ofmouse, that is, 129/Sv and C57BL/6J, were mixed. Therefore, a pure linemouse was sufficiently backcrossed (4 generations) to be suitable foranalyzing the physiological function of PTPζ, and a PTPζ gene-deficientmouse from which the influence of other genes was eliminated wasgenerated. By comparative analysis of a mouse whose function of gene DNAthat encodes the PTPζ was deficient on its chromosome and a wild typemouse, it is revealed for the first time that the PTPζ gene-deficientmouse, which has grown and propagated normally, and where no majormorphologic abnormality has been identified, has dysfunction of centralmonoamine pathway, such as changes in the level of central monoaminemetabolism, hyposensitivity to a stimulant drug (methamphetamine),dysfunction of mesolimbic dopamine pathway, delay in acclimating to newcircumstances and increase in stress-responsiveness. Further, it wasfound that PTPζ, which is thought to be a receptor of VacA, a toxin ofHelicobacter pylori, is expressed in gastric epithelial cell layer of amouse. The present invention has thus been completed.

DISCLOSURE OF THE INVENTION

[0007] The present invention relates to a method for screening a PTPζinhibitor or activator wherein a subject material is administered to anon-human animal whose function of gene DNA that encodesproteoglycan-type receptor-type protein tyrosine phosphatase (PTPζ) isdeficient on its chromosome and a wild-type non-human animal, and PTPζactivities in these non-human animals are compared and evaluated (claim1), the method for screening a PTPζ inhibitor or activator according toclaim 1, wherein the comparison and the evaluation of PTPζ activity isthe comparison and the evaluation of the function of central monoaminepathway (claim 2), the method for screening a PTPζ inhibitor oractivator according to claim 2, wherein the comparison and theevaluation of the function of central monoamine pathway is thecomparison and the evaluation of changes in the level of centralmonoamine metabolism, sensitivity to a stimulant drug, the presence ofdysfunction of mesolimbic dopamine pathway, level of acclimation to newcircumstances, or stress-responsiveness (claim 3), the method forscreening a PTPζ inhibitor or activator according to claim 1, whereinthe comparison and the evaluation of PTPζ activity is the comparison andthe evaluation of the level of binding to VacA, a tokin of Helicobacterpylori (claim 4), the method for screening a PTPζ inhibitor or activatoraccording to claim 1, wherein the comparison and the evaluation of PTPζactivity is the comparison and the evaluation of the level of binding topleiotrophin, a heparin-binding secretory protein (claim 5), the methodfor screening a PTPζ inhibitor or activator according to any one ofclaims 1 to 5, wherein the non-human animal whose function of gene DNAthat encodes PTPζ is deficient on its chromosome is purified by beingbackcrossed for 4 or more generations (claim 6), the method forscreening a PTPζ inhibitor or activator according to any one of claims 1to 6, wherein the non-human animal is a mouse (claim 7), a PTPζinhibitor or activator obtained by the method for screening aproteoglycan-type receptor-type protein tyrosine phosphatase (PTPζ)inhibitor or activator according to any one of claims 1 to 7 (claim 8),the PTPζ inhibitor or activator according to claim 8, wherein the PTPζinhibitor or activator is a binding inhibitor between PTPζ and VacA, atoxin of Helicobacter pylori (claim 9), the PTPζ inhibitor or activatoraccording to claim 8, wherein the PTPζ inhibitor or activator is abinding inhibitor between PTPζ and pleiotrophin, a heparin-bindingsecretory protein (claim 10), a remedy for dysfunction of centralmonoamine pathway containing the proteoglycan-type receptor-type proteintyrosine phosphatase (PTPζ) inhibitor or activator according to claim 8as an active component (claim 11), a remedy for gastric ulcer orgastritis caused by Helicobacter pylori containing the proteoglycan-typereceptor-type protein tyrosine phosphatase (PTPζ) inhibitor according toclaim 8 or 9 as an active component (claim 12), a remedy for gastriculcer or gastritis caused by pleiotrophin containing theproteoglycan-type receptor-type protein tyrosine phosphatase (PTPζ)inhibitor according to claim 8 or 10 as an active component (claim 13).

[0008] The present invention also relates to a non-human model animalwhose function of gene DNA that encodes proteoglycan-type receptor-typeprotein tyrosine phosphatase (PTPζ) is deficient on its chromosome andwhich is hyposensitive to a central stimulant drug (an addictive drug)(claim 14), the non-human model animal according to claim 14, whereinthe non-human animal is hyposensitive to a stimulant drug (claim 15),the non-human model animal according to claim 14 or 15, wherein thenon-human animal whose function of gene DNA that encodes PTPζ isdeficient on its chromosome is purified by being backcrossed for 4 ormore generations (claim 16), the non-human model animal according to anyone of claims 14 to 16, wherein the non-human animal is a mouse (claim17), a non-human model animal whose function of gene DNA that encodesproteoglycan-type receptor-type protein tyrosine phosphatase (PTPζ) isdeficient on its chromosome and which is hyposensitive to VacA, a toxinof Helicobacter pylori (claim 18), a non-human model animal whosefunction of gene DNA that encodes proteoglycan-type receptor-typeprotein tyrosine phosphatase (PTPζ) is deficient on its chromosome andwhich is hyposensitive to pleiotrophin, a heparin-binding secretoryprotein (claim 19), the non-human model animal according to claim 18 or19, wherein the non-human animal whose function of gene DNA that encodesPTPζ is deficient on its chromosome is purified by being backcrossed for4 or more generations (claim 20), and the non-human model animalaccording to any one of claims 18 to 20, wherein the non-human animal isa mouse (claim 21).

BRIEF EXPLANATION OF THE DRAWINGS

[0009]FIG. 1 is a view showing the examination results of characterphenotypes of PTPζ-deficient mice in an open field test and circadianrhythm.

[0010]FIG. 2 is a view showing the changes of monoamine metabolism inthe brains of PTPζ-deficient mice.

[0011]FIG. 3 is a view showing the examination results ofimmunohistochemistry of dopamine pathway in PTPζ-deficient mice.

[0012]FIG. 4 is a view showing the decrease of locomotor activity tomethamphetamine and GBR 129909 in PTPζ-deficient mice.

[0013]FIG. 5 is a view showing the abnormal DA neurotransmission innucleus accumbens of PTPζ-deficient mice.

[0014]FIG. 6 is a view showing the examination results of expressionproperty of PTPζ in dopamine pathway of adult mice brains.

[0015]FIG. 7 is a view showing the results of stress and fear behaviorsin PTPζ-deficient mice.

[0016]FIG. 8 is a view showing the results of exploration behavior ofwild-type mice and PTPζ-deficient mice to a novel object.

[0017]FIG. 9 is a view showing the examination results of PTPζexpression in gastric epithelial cell layer of wild-type mice andPTPζ-deficient mice.

[0018]FIG. 10 is a view showing the examination results of transcription(RT-PCR) and expression (Western blot) of PTPζ in gastric epithelialcell layer of mice.

[0019]FIG. 11 is a view showing the results of gastric ulcer formationin wild-type mice and PTPζ-deficient mice orally administered with VacA,a toxin of Helicobacter pylori.

[0020]FIG. 12 is a view showing the examination results of tyrosinephosphorylation of GIT1, a substrate molecule of PTPζ, caused bystimulus with VacA.

[0021]FIG. 13 is a view showing the results of mucosal damage in gastricepithelium of mice caused by administration of pleiotrophin.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The method for screening a PTPζ inhibitor or activator accordingto the present invention is not particularly limited as long as it is amethod for screening wherein a subject material is administered to anon-human animal whose function of gene DNA that encodes PTPζ isdeficient on its chromosome and a wild-type non-human animal, and PTPζactivities in these non-human animals are compared and evaluated(analyzed). An example of the non-human animal whose function of geneDNA that encodes PTPζ is deficient on its chromosome includes anon-human animal wherein a part or the whole of its endogenous gene thatencodes PTPζ is inactivated by gene mutation such as disruption,deficiency, substitution or the like, and the function of expressingPTPζ in wild type is lost. In addition, as the non-human animalaccording to the present invention, rodents including a mouse, a rat anda guinea pig are specifically exemplified, but not limited to theseanimals. The method for constructing a non-human animal whose functionof gene that encodes PTPζ is deficient on its chromosome is explainedbelow with an example of a mouse whose function of PTPζ gene isdeficient on its chromosome.

[0023] A mouse whose function of PTPζ gene is deficient on itschromosome, in other words, a PTPζ knockout mouse can be constructed bythe method described in the aforementioned paper written by the presentinventors (Neuroscience Letters 274, 135-138, 1998), or other suchmethods. Specifically, the construction method comprises the steps of: aPTPζ gene is screened by using a gene fragment obtained from mouse genelibrary through PCR or the like; a part or the whole of the screenedPTPζ gene is substituted with a marker gene, for example, a Lac-Z gene,a neomycin-resistant gene or the like, and if necessary, a gene such asa diphthelia toxin A fragment (DT-A) gene or a herpes simplex virusthymidine kinase (HSV-tk) gene is introduced into 5′-terminal side, toconstruct a targeting vector; this constructed targeting vector islinearized and introduced into an ES cell by microinjection,electroporation or other such methods and then homologously recombined;among the homologous recombinants, an ES cell resistant to X-galstaining or antibiotics such as G418, ganciclovir (GANC) or the like isselected. It is preferable to confirm whether the selected ES cell isthe intended recombinant by Southern blotting or other such methods.

[0024] The above-mentioned recombined ES cell is microinjected into amouse blastocyst, and then the blastocyst is transplanted into arecipient to generate a chimeric mouse. A heterozygous mouse can beobtained by intercrossing the chimeric mouse and a wild-type mouse, anda PTPζ knockout mouse can be obtained by intercrossing the heterozygousmice. Further, by backcrossing the obtained knockout mouse with a pureline mouse, PTPζ knockout mice whose genetic background other than PTPζis in uniform can be obtained. Any pure line mouse can be used as thepure line mouse, for instance, C57BL/6J is specifically exemplified.Examples of the method for confirming whether PTPζ is present in thePTPζ knockout mouse include; Southern blotting or the like of DNAseparated from a part of a tail tip of a mouse obtained by theabove-mentioned methods, Northern blotting or the like of RNA isolatedfrom neurons or the like of the mouse, and Western blotting or the likeof PTPζ expression in the mouse.

[0025] As the method for screening a PTPζ inhibitor or activatoraccording to the present invention, a method wherein a subject materialis respectively administered to a non-human animal whose function ofgene that encodes PTPζ is deficient on its chromosome, for example, aPTPζ knockout mouse, and a wild-type mouse, preferably a wild-typelittermate, and PTPζ activities in the PTPζ knockout mouse and in thewild-type mouse are compared and evaluated is exemplified. Examples ofthe comparison and the evaluation of the PTPζ activities mentioned aboveare, the comparison and the evaluation of the function of centralmonoamine pathway including the comparison and the evaluation of changesin the level of central monoamine metabolism, sensitivity to a stimulantdrug, the presence of dysfunction of mesolimbic dopamine pathway, levelof acclimation to new circumstances or stress-responsiveness; thecomparison and the evaluation of the level of binding to VacA, a toxinof Helicobacter pylori, particularly the level of binding inhibitionbetween PTPζ and VacA, a toxin of Helicobacter pylori, and thecomparison and the evaluation of the level of binding to pleiotrophin, aheparin-binding secretory protein, particularly the level of bindinginhibition between PTPζ and pleiotrophin, a heparin-binding secretoryprotein, that are explained in detail in the examples.

[0026] The method for administering a subject material in the method forscreening a PTPζ inhibitor or activator is not particularly limited, andoral or intravenous administration or the like can be used. In suchmethod, after administering a subject material, PTPζ activities in thePTPζ knockout mouse and in the wild-type mouse are measured, and thencompared and evaluated. The higher cerebral function in which monoaminepathway is involved varies widely, and it is known that its dysfunctionand degeneration lead to various disease states. Consequently, in thecase where dysfunction of central monoamine pathway in a PTPζ knockoutmouse is ameliorated/cured to the same level as the function of awild-type mouse, the subject material is thought to be a PTPζ activatorsuch as a PTPζ agonist, and the PTPζ activator shows promise as a remedyfor dysfunction of central monoamine pathway, for example, as a novelremedy not only for Parkinson's disease or Huntington's disease, whichare serious neurodegenerative diseases, autism or the like, but also fornervous affections including manic-depression, attention-deficitdisorder, and drug addiction. In addition, there is a possibility thatthe PTPζ inhibitor discovered by the comparison and the evaluation ofthe function of central monoamine pathway is useful as a remedy forgastric ulcer and gastritis caused by VacA, a toxin of Helicobacterpylori, or pleiotrophin, a heparin-binding secretory protein mentionedbelow.

[0027] VacA, an exotoxin secreted by Helicobacter pylori which is wellknown as a cause of gastric ulcer, is detected in about 90% or more ofthe patients who suffer from acute gastritis or gastric ulcer, and it isknown that a mouse orally administered with VacA, a toxinextracellularly secreted by Helicobacter pylori, shows the onset ofacute gastritis. Consequently, in the case where the level of binding toVacA, a toxin of Helicobacter pylori, in a wild-type mouse isameliorated to a same level as that in a PTPζ knockout mouse, it meansthat the subject material is a PTPζ inhibitor such as a PTPζ antagonistand a binding inhibitor between PTPζ and VacA, a toxin of Helicobacterpylori, and the PTPζ inhibiting substance/PTPζ inhibitor shows promiseas a novel remedy for gastric ulcer and gastritis. There is apossibility that the above-mentioned PTPζ activator discovered by thecomparison and the evaluation of the level of binding to VacA, a toxinof Helicobacter-pylori, is useful as a remedy for dysfunction of centralmonoamine pathway. The present inventors have revealed for the firsttime that pleiotrophin causes gastric ulcer by binding to PTPζ, and theinvolvement of pleiotrophin in the formation of gastric ulcer has neverbeen known. A PTPζ-deficient mouse is a negative control of theformation of gastric ulcer caused by pleiotrophin.

[0028] Further, the present inventors have revealed for the first timethat 18 kD pleiotrophin, a heparin-binding secretory protein, causesgastric ulcer by binding to PTPζ. Therefore, in the case where the levelof binding to pleiotrophin in a wild-type mouse is ameliorated to a samelevel as that in a PTPζ knockout mouse it means that the subjectmaterial is a PTPζ inhibitor such as a PTPζ antagonist and a bindinginhibitor between PTPζ and pleiotrophin, and the PTPζ inhibitingsubstance/PTPζ inhibitor shows promise as a novel remedy for gastriculcer and gastritis. There is a possibility that the above-mentionedPTPζ activator discovered by the comparison and the evaluation of thelevel of binding to plelotrophin is useful as a remedy for dysfunctionof central monoamine pathway.

[0029] The non-human model animal hyposensitive to a stimulant drug (anaddictive drug) according to the present invention is not particularlylimited as long as it is a PTPζ knockout mouse whose function of geneDNA that encodes proteoglycan-type receptor-type protein tyrosinephosphatase (PTPζ) is deficient on its chromosome, or the like, but aknockout backcrossed for 4 or more generations is preferable. Theusefulness of a PTPζ knockout mouse, in particular, its hyposensitivityto a stimulant drug (an addictive drug) had not been known until it wasrevealed for the first time by the present inventors. The term“hyposensitivity” here means lower level of reactivity to a stimulantdrug in comparison to the reactivity level of wild-type non-humananimals. The non-human model animal hyposensitive to a stimulant drug(an addictive drug) according to the present invention includes thosefrom slightly hyposensitive ones to totally unresponsive ones. Inaddition, as the stimulant drug (the addictive drug), methamphetamine,amphetamine that has a same effect as methamphetamine, cocaine andcannabis that have a same effect as dopamine transporter inhibitor GBR12909, LSD that has a same effect as p-chloro-amphetamine, which is aserotonin liberator, selective serotonin reuptake inhibitor (SSRI) usedas an antidepressant such as fluoxetine, fluoxamine, paroxetine,serrtraline or the like, are exemplified. The non-human model animalhyposensitive to a central stimulant drug (an addictive drug) accordingto the present invention is hyposensitive to a stimulant drug (anaddictive drug), and in addition, shows abnormal neurological propertiesincluding the changes in the level of central monoamine metabolism,dysfunction of mesolimbic dopamine pathway, delay in acclimating to newcircumstances or increase in stress-responsiveness, as described indetail in examples mentioned below. Consequently, the non-human modelanimal is not only useful as an experimental model animal forelucidating a signal transmission mechanism in the central nervoussystem such as a mechanism of response to a central stimulant drug (anaddictive drug), but also available for screenings of novel remedies fordrug addiction or nervous affections as mentioned above.

[0030] The non-human model animal hyposensitive to VacA which is a toxinof Helicobacter pylori or pleiotrophin which is a heparin-bindingsecretory protein according to the present invention is not particularlylimited as long as it is a PTPζ knockout mouse whose function of geneDNA that encodes proteoglycan-type receptor-type protein tyrosinephosphatase (PTPζ) is deficient on its chromosome, or the like, but aknockout mouse backcrossed for 4 or more generations is preferable. Theusefulness of a PTPζ knockout mouse, in particular, its hyposensitivityto VacA which is a toxin of Helicobacter pylori or pleiotrophin which isa heparin-binding secretory protein had not been known until it wasrevealed by the present invention. The non-human model animalhyposensitive to VacA which is a toxin of Helicobacter pylori orpleiotrophin which is a heparin-binding secretory protein according tothe present invention is not only useful as an experimental model animalfor elucidating the onset mechanism of gastric ulcer caused by VacAwhich is a toxin of Helicobacter pylori or pleiotrophin which is aheparin-binding secretory protein, but also available as an advantageousnegative control for the screening of a novel remedy for gastric ulceras mentioned above.

[0031] The present invention is explained below more specifically withreference to examples, however, the present invention is not limited tothese examples. Example 1 (Physiological role of PTPζ in centraldopamine pathway and monoamine pathway, and usefulness of aPTPζ-deficient mouse).

[0032] Though large amount of PTPζ is expressed in the central nervoussystem, its neurophysiologic role has been unknown. Therefore, theimportance of PTPζ molecule in higher cerebral function was examined atindividual level by comparing and analyzing a PTPζ gene-deficient mouseand a wild-type mouse behaviorally and neuropharmacologically.

EXAMPLE 1-1

[0033] [Method]

[0034] (A-1 Animals)

[0035] A PTPζ-deficient mouse was generated by the method described inthe aforementioned paper written by the present inventors (NeuroscienceLetters 274, 135-138, 1998). A LacZ gene was inserted into the positionimmediately after the translation initiation codon in exon 1 of a PTPζgene of a mutant mouse and accordingly, a LacZ gene was expressed underthe control of expression regulatory unit of the PTPζ gene. In thisexperiment, a knockout mouse was backcrossed with inbred C57BL/6J linefor 4 generations, and male littermates of 2 to 5 months old were used.The animals were housed in an animal care facility at 25.degrees C.,with a 12/12 hours light-dark cycle and fed food and water ad libitum.Animals were cared in accordance with the institutional guidelines.

[0036] (A-2 Behavioral Experiment)

[0037] A behavioral experiment was conducted during the light cycle from7:00 a.m. to 7:00 p.m.

[0038] A-2-1

[0039] In an open field test, a mouse was placed in the center of a grayround field (internal diameter: 80 cm, height: 40 cm) divided into 25equal segments by grids. Locomotor activity was measured as the numberof line crossings of the grid during 5 minutes.

[0040] A-2-2

[0041] Circadian motility was measured with an infrared ray passivesensor system (AB system 24A, Neuroscience, Inc.). For the measurement,a sensor was set on the top of a normal mouse cage (30×20×13 cm) wherethe animals were housed individually with ad libitum food and water for7 days. Analysis and evaluation were made with the measured data of thelast 3 days.

[0042] A-2-3

[0043] In a forced swimming test, a mouse was forced to swim for 15minutes in 2 consecutive days in a cylindrical container (diameter: 8cm, height: 20 cm) which contained water at 22. degree. C. to a depth of8 cm. Locomotion was measured every 5 minutes with an infraredmonitoring apparatus (SCANET MV-10, Toyo Sangyo Co. Ltd.).

[0044] A-2-4

[0045] An elevated plus maze consisted of a central area (6×6 cm) and 4arms placed 50 cm above floor. Two “closed arms” were enclosed withinwalls (30 cm), and the other two “open arms had low rims (1 cm). A mousewas placed in the center of the maze and the number of entries and timespent in the open and closed arms were counted for 5 minutes under a dimlight condition (about 80 lux).

[0046] A-2-5

[0047] An exploration behavior to a novel object was evaluated with anopen field container and a plastic cube (9 cm on a side). On the daybefore the experiment, a mouse was placed in an open field for about 10minutes to be acclimated to the circumstance in advance. On the day ofthe experiment, the mouse was placed in the field that containednothing, and observed for 9 minutes. Then, a cube-shaped plastic blockwas placed quietly in the center of the central area of the field, andthe mouse was further observed for 9 minutes. As to the result of theobservation, the number of times that the mouse crossed the partitionlines in the open field was counted and evaluated as mobility, and thenumber of times that the mouse crossed the partition lines in the wholearea of the field and a certain area in the center of the field wereevaluated respectively.

[0048] (A-3 Pharmacological Experiments)

[0049] The effects of psychostimulant drugs on locomotor activity weremeasured in a clear acrylic chamber (40×40×40 cm) by measuring animalmovements with an electromagnetic activity monitoring system (sensorunit 0603, scanner 1099, Panlab). Before drug administration, theanimals were acclimated to the measurement circumstance for at least 90minutes. The animals were then administered with a stimulant drug,methamphetamine (METH) (1 mg/kg, s.c.), GBR12909 (2 mg/kg, i.p.),apomorphine (1 mg/kg, s.c.), or p-chloro-amphetamine (2 mg/kg, i.p.),and saline as administered to a control, and locomotor activity wasmonitored for the next 60 minutes.

[0050] (A-4-1 Quantification of Dopamine and its Metabolites)

[0051] Brain tissues were dissected on ice and extracted by sonicationin a solution containing 10 mM HClO₄, 0.1 mM sodium pyrosulfite, 20 μMEDTA-2Na and 10 pg/ml (±) isoproterenol as an internal standard, and theextract was centrifuged at 15,000 rpm for 10 minutes. Dopamine and itsmetabolites in the supernatant were measured by automated HPLC with anelectrochemical detector (Coulochem II, MC Medical, Inc.) under thefollowing condition. The separation was performed on MCM C-18 column(4.6×150 mm, MC Medical, Inc.) using 50 mM acetate-citrate buffer (pH3.0) containing 3.1% acetonitrile, 7.6% methanol, 4.4 mM sodium1-heptanesulfonate, and 0.1 mM EDTA.2Na, at a flow rate of 1.0 ml/min ina column-chamber maintained at 37.degree. C. The working electrode wasset at +450 mV.

[0052] (A-4-2 In Vivo Microdialysis)

[0053] Mice were anaesthetized with sodium pentobarbital (80 mg/kg i.p.)and placed in a stereotaxic apparatus. A hole was drilled through theskull and an intracerebral guide cannula (CMA 11, CMA/Microdialysis AB)was inserted into the nucleus accumbens (NAC) and fixed with dentalcement. The stereotaxic coordinates for implantation of the guidecannula in both wild-type and PTPζ-deficient mice were: anterior, +1.1mm, ventral, +3.6 mm, lateral, +1.0 mm to the surface of bregma. 24hours after the surgery, mice were chained to a counterbalance lever armattached to a locomotor activity chamber (30×30 cm) with an infraredmonitoring apparatus (SCANET LC-10, Toyo Sangyo Co., Ltd.). Dialysisprobes (membrane length 1 mm; external diameter 0.24 mm; cuprophane CUP11, CMA/Microdialysis AB) were inserted through the guide cannula andperfusion was performed with artificial cerebrospinal fluid (ACSF), pH7.4, (in mM; 145.ONaCl, 2.7 KCl, 1.2 CaCl₂, 1.0 MgCl₂, and 2.0 NaH₂PO₄)at a flow rate of 2 μl/min. Perfusates were collected from freely movinganimals every 20 minutes in a tube containing 20 μl of 0.4 M HClO₄ (CMA170). Samples were collected and assayed using HPLC-ECD as describedabove except for the following modifications: the column was MCM C-18(4.6×150 mm) and the buffer contained 75 mM NaH₂PO₄, 1.7 mM1-octanesulfonic acid sodium salt, 0.1 mM triethylamine, 25 μM EDTA-2Naand 10% acetonitrile, at flow rate of 0.55 ml/min at 30.degree. C. Theelectrode was set at +320 mV. METH administration (1 mg/kg s.c.) orlocal infusion of high K⁺ solution (in mM: 47.7 NaCl, 100.0 KCl, 1.2CaCl₂, 1.0 MgCl₂, and 2.0 NaH₂PO₄, pH 7.4) through the microdialysisprobe was performed after the state became steady.

[0054] (A-5 Immunohistochemistry)

[0055] Mice were anaesthetized with sodium pentobarbital and perfusedand fixed with 4% paraformaldehyde solution. Brains were dissected andincubated overnight in 0.1 M phosphate buffer (PB) containing 30%sucrose at 4.degree. C. The brains were cut into 40 μm sections on acryostat. The sections were treated with 20 mM PB-saline (PBS)containing 3% H₂O and 0.05% NP-40 for 30 minutes, and then blocked with10% normal goat serum and 0.05% TritonX-100 in PBS. The sections werethen incubated overnight with rabbit anti-tyrosine hydroxylase (1:1000)(AB152, Chemicon International Inc.). The binding of specific antibodieswas detected by using an ABC peroxidase kit (Vectors Laboratories, Inc.)according to the manufacture's instructions with 3,3-diaminobenzidine(DAB) and H₂O₂ as the substrate. To detect the β-galactosidase whichreflect the PTPζ-gene expression, tissue sections from the heterozygousmutant mice were subjected to X-gal staining before immunohistochemicalanalyses (Neuroscience Letters 274, 135-138, 1998).

[0056] (A-6 Dopamine Uptake Assay)

[0057] Dopamine (DA) uptake was measured as follows. Striatalsynaptosome tissue pools (3 animals/pool) were homogenized in 100 volume(v/w) of 0.32 M sucrose in a teflonglass homogenizer and centrifuged at12,000 rpm for 10 minutes. The crude synaptosomal pellet was resuspendedin 0.32 M sucrose at a concentration of 30 mg/ml (original wet weight)and divided into 30 μl aliquots. Each aliquot was added with 270 μl ofKrebs-Ringer phosphate buffer with increasing concentrations of [³H] DA(final 1-10 nM, 88.5 Ci/nmol, Amersham Pharmacia Biotech UK Ltd.). Afterincubating the resultant solution for 3 minutes at 30.degree. C., uptakewas quickly terminated by filtration through Whatman GF/C glass filters.The filters were rinsed three times with 2.5 ml ice-cold 0.32 M sucroseand measured by using a liquid scintillation counter at an efficiency of50%. Blank values were measured in the presence of 10 μM GBR12909.

[0058] (A-7 Synthesis of cDNA from a Single Cell)

[0059] The cDNA preparation from single cells was carried out asdescribed previously (J. Neuroscience 18, 3124-3137, 1998). Mice brainswere removed quickly after decapitation, and cut into 400 μm thickcoronal midbrain slices with a microslicer while bathed in low Ca²⁺HEPES-buffered saline (in mM: 140.0 Na isethionate, 2.0 KCl, 4.0 MgCl₂,0.1 CaCl₂, 23.0 glucose, and 15.0 HEPES). The slices were maintained forone hour at room temperature in NaHCO₃ buffered saline (having 126.0NaCl, 2.5 KCl, 2.0 MgCl₂, 26.0 NaHCO₃, 1.25 NaH₂PO₄, 1.0 pyruvic acid,0.2 ascorbic acid, 0.1 NG-nitro-L-arginine, 1.0 kynurenic acid, and 10.0glucose, adjusted to be pH 7.4 with NaOH, in mM). The ventral tegmentalarea and substantia nigra were cut out and put in low Ca²⁺, and thentreated with 1 mg/ml pronase in HEPES-buffered HBSS at 35.degree. C. for30 minutes in an oxygenated Cell-stir chamber (Wheaton, Inc.). Thetissue was rinsed three times with low Ca²⁺ HEPES-buffered saline andits cells were dissociated by repeated pipetting with Pasteur pipettes.The cell suspension was poured into a 35 mm Lux Petri dish containingHEPES-buffered HBSS, which was mounted on the stage of a microscope.After allowing cells to settle down, the solution was replaced by HEPESbuffer. The content of the cells were aspirated into an electrodepipette filled with diethyl pyrocarbonate (DEPC)-treated water. Thecontent was immediately recovered and added to a mixture of 5 μlDEPC-treated water (H₂O), 0.5 μl RNAsin (28,000 U/ml, PromegaCorporation), 0.5 μl dithiothreitol (DTT, 0.1 M), and 0.1 μl randomhexamer primer (50 ng/μl). The resulting mixture was heated at70.degree. C. for 10 minutes, and then iced quickly for 5 minutes. ThecDNA was synthesized by adding 1 μl of Superscript II reversetranscriptase (200 U/μl) (Life Technologies), 4 μl of 5xreaction buffer,and 1 μl of 10 mM dNTP to cell mRNA. After conducting the reaction in atotal volume of 20 μl at 42.degree. C. for 50 minutes, a treatment wasperformed at 70.degree. C. for 15 minutes.

[0060] (A-8 Multiplex and Nested PCR)

[0061] Multiplex and nested PCR were performed as previously described(EMBO J. 18, 833-846, 1999) with modifications. Single-cell cDNA wastreated with RNase (1 μl, 2 U/μl) at 37.degree. C. for 20 minutes. Theprimer sets of marker genes, tyrosine hydroxylase (TH, GenBank accessionNo. M69200), glutamic acid decarboxylase 67 (GAD67, accession No.Z49976), and glial acidic fiber protein (GFAP, accession No. K01347),were reported by Liss et al. (EMBO J. 18, 833-846, 1999), and the primersets to amplify PTPζ (accession No. U09357) were constructed in thisexperiment. The cDNA of TH, GAD67, GFAP, and PTPζ were amplifiedsimultaneously in a single tube by multiplex PCR using the first primermixture (for PTPζ from 5′ to 3′; sense [Seq. ID. No. 1]:GGTCCACTGAAGTCCACAGC; position 5512 to 5531, antisense [Seq. ID. No. 2]:TCT AGT ACA ATG TAT GTG CCC G; position 5948 to 5927). The initialmultiplex-PCR was performed in a final volume of 20 μl containing 5 μlof the single-cell cDNA, 20 pmol of each primer, 200 μM dNTPs, 2 μl of10× PCR buffer, and 1 U of EX-Taq (Takara Shuzo, Co., Ltd.). The PCR wasconducted with a Thermal Cycler MP (Takara) under the followingconditions: 94.degree. C. for 5 minutes followed by 25 cycles at95.degree. C. for 30 seconds, 62.degree. C. for 3 seconds, and72.degree. C. for 2 minutes, with a treatment at 72.degree. C. for 5minutes. The nested-PCR was carried out in four individual reactions ina final volume of 20 μl containing 0.1 μl of the initial PCR product,200 μM dNTPs, 2 μl of 10×PCR buffer, 0.5 U of EX-Taq, and 10 pmol ofeach of primer pairs (for PTPζ sense [Seq. ID. No. 3]: CGG GAG CTT CCTGGT CAA CCA G; position 5655 to 5677, antisense [Seq. ID. No. 4]: AGCACG GGT AGG GAG TAC TC; position 5873 to 5824). To investigate thepresence and the size of the amplified fragments, 5 μl aliquots of thePCR products were separated by electrophoresis on 3% Nusive 3:1 agarosegel (FMC BioProducts, Rockland), and visualized with ethidium bromidestaining. The predicted sizes (bp) of the eight PCR-generated fragmentswere: 189 (PTPζ), 377 (TH), 517 (GFAP), and 702 (GAD67). The primers forPTPζ can only amplify the intracellular phosphatase domain D1, which ispresent only in the sequences of the PTPζ-A and -B isoforms, but not inthe PTPζ-S which is also known as phosphacan (J. Biochem. (Tokyo), 123,458-467, 1998).

EXAMPLE 1-2

[0062] [Results]

[0063] (Behavioral Phenotype of PTPζ-Deficient Mice)

[0064] In order to elucidate the behavioral phenotype of PTPζ-deficientmice, behavioral observation was conducted by an open field test (theabove-mentionedmethods A-1 and A-2-1). Locomotor activity (A) andrearing (B) of wild-type mice (PTPζ^(+/+), n=10) and PTPζ-deficient mice(PTPζ^(−/−), n=12) were measured for consecutive 2 days by the openfield test, and the results are shown in FIGS. 1A and 1B, respectively.The data are presented as mean±SEM (*p<0.05). As a result,PTPζ-deficient mice exhibited larger amount of locomotor activity thanwild-type mice, and significantly high motility in the open field on thefirst day. However, 24 hours later, the change disappeared in the test(FIG. 1A). On the other hand, significant change was not found in valuesfor rearing, an exploration behavior (FIG. 1B). It was revealed that thePTPζ-deficient mice increased their responsibility of locomotor activityto novel circumstances.

[0065] Locomotor activity in home-cage condition was examined forconsecutive days (the above-mentioned method A-2-2). Mice were housedindividually for 7 days, and locomotor activity was analyzed with aninfrared ray passive sensor system during the last 3 days (PTPζ^(+/+),n=14 and PTPζ^(−/−), n=14). The results are shown in FIG. 1C. The dataare presented as mean+SEM per each 30 minutes interval (*p <0.05). ThePTPζ^(−/−) mice showed a marked decrease in the peak of circadianactivity during the early dark phase compared with the wild-type mice,and their activity peak immediately after lights were put off (19:00 to24:00) was significantly low in comparison with the wild-type mice,though their activity rhythm was normal.

[0066] (Abnormal Metabolism of Monoamine in Brain)

[0067] It has been known that locomotor activity and circadian rhythmare deeply linked to monoaminergic systems in the brain. Therefore,tissue contents of dopamine (DA), norepinephrine (NE), serotonin (5-HT)and their metabolites (DOPAC, HVA, metabolites of DA; MHPG, a metaboliteof NE; 5-HIAA, a metabolite of 5-HT) in the brains were measured byhigh-performance liquid chromatography (HPLC) and an electrochemicaldetector (ECD) (the above-mentioned method A-4-1). The results are shownin FIG. 2. The data are presented as mean±SEM from PTPζ^(+/+) mice (n=7)and PTPζ^(−/−) mice (n=7) (*p<0.05, **p<0.01). As a result, metabolismof monoamine in the brains of the PTPζ-deficient mice was revealed to beapparently abnormal. There was almost no difference between the twogenotypes in the tissue content of DA (A). However, the ratios ofDOPAC/DA (B) and HVA/DA (C), 5-HIAA/5-HT (G) in several brain regions ofthe PTPζ-deficient mice were markedly decreased compared to those of thewild-type mice. The content of NE in the PTPζ-deficient mice increasedcompared to that of the wild-type mice.

[0068] (Immunohistological Analysis of DA Pathway)

[0069] Next, DA pathway of PTPζ-deficient mice was analyzedimmunohistologically for detecting the presence of histological changetherein with a specific antibody to tyrosine hydroxylase, a markerenzyme of DA neurons (the method A-5). The results are shown in FIG. 3.Regions of wild-type mice (+/+, left side) and PTPζ-deficient mice (−/−,right side) through the substantia nigra and ventral tegmental area(low-magnification photographs, a and b; and high-magnificationphotographs, c and d), and striatum (e and f) were analyzedimmunohistochemically with polyclonal antibodies specific to tyrosinehydroxylase (TH). According to the results, no significant differencewas identified between DA pathways of the PTPζ-deficient mice and thewild-type mice, and DA pathway was considered to remainimmunohistologically normal after PTPζ became deficient. The scale bars,500 μm (a, b, e, and f), and 50 μm (c and d).

[0070] (Decrease in Sensitivity to Stimulant Drugs)

[0071] Methamphetamine (METH), a stimulant drug, acts on DA pathway andincreases the locomotion of mice markedly. In expectation of apossibility of functional change in DA pathway of PTPζ-deficient mice,sensitivity to METH was analyzed by an electromagnetic locomotionmeasuring apparatus (the above-mentioned method A-3). The results areshown in FIG. 4. Mice were placed in an acrylic chamber, and locomotionwas measured by the counts of electromagnetic sensors. PTPζ^(−/−) mice(n=9) and PTPζ^(+/+) mice (n=9) showed similar time course of locomotoractivity before drug administrations. After an adequate acclimation (atleast 90 minutes), methamphetamine (METH, 1 mg/kg, s. c.) or saline(arrow point) was injected, and it was indicated that the PTPζ^(−/−)mice exhibited a significantly attenuated locomotor response compared tothe wild-type mice (FIG. 4A). No change was observed in the controlgroup administered with saline. The data are presented as mean±SEM pereach 20 minutes interval (p<0.001 for phenotype).

[0072] Effects of METH (1 mg/kg, s. c.), GBR12909 (2 mg/kg, i. p.),apomorphine (2 mg/kg, s. c.), p-chloro-amphetamine (2 mg/kg, i. p.) andsaline on locomotor activity are presented in FIG. 4B as the totalcounts of locomotion during the 60 minutes interval after drugadministration. Locomotor activity was measured in the same manner asabove-mentioned. The data are presented as mean±SEM (*p<0.05, **p<0.01).PTPζ-deficient mice showed similar hyposensitivity also to GBR12909, aspecific inhibitor of DAT (FIG. 4B). On the other hand, thoughapomorphine, a non-specific agonist for DA receptors, shows similareffect by stimulating a postsynaptic neuron, responsiveness toapomorphine was normal (FIG. 4B). These results suggest thatneurotransmitting function of a presynaptic neuron is abnormal in DApathway of the PTPζ-deficient mice. The PTPζ-deficient mice werehyposensitive also to p-chloro-amphetamine that liberates 5-HT from apresynaptic region (FIG. 4B).

[0073] [³H] DA was mixed with striatal synaptosomes and [³H] DA uptakeinto tissues was measured (the above-mentioned method A-6-1). Thehomogenate of the striatal synaptosomes was incubated for 2 minutes at30.degree. C. in the presence of [³H] DA (1 to 10 nM). The amount of DAuptake was analyzed by Eadie-Hofstee plot, and maximum velocity (Vmax,pmol/2 min/mg protein) and affinity (Km, nM) are presented in Table 1,as mean±SEM of three measurement values (*P<0.05). From Table 1, it wasfound that both of maximum velocity (Vmax) and affinity (Km) of DAuptake amount are changed dynamically, though there was no difference inthe number of DAT. TABLE 1 [³H] DA uptake Mice Km (nM) Vmax (pmol/3min/mg) PTPζ^(+/+) 109.2 ± 2.45 45.74 ± 2.77 PTPζ^(−/−) 147.3 ± 11.358.70 ± 1.03

[0074] (Abnormality of DA Neurotransmission in Nucleus Accumbens)

[0075] Effect of activating locomotor activity caused by administrationof METH or GBR12909 is induced mainly by an increase of extracellular DAconcentration in nucleus accumbens (NAC). The change of extracellular DAconcentration in nucleus accumbens after the administration of METH wasanalyzed by microdialysis (the above-mentioned method A-4-2).METH-induced DA release in the NAC was analyzed by microdialysis, andthe results are shown in FIG. 5A. Each value is mean±SEM from PTPζ^(+/+)mice (n=7) and PTPζ^(−/−) mice (n=6), and the precentage value to theaverage of 2 samples before METH stimulation. The PTPζ-deficient miceexhibited a markedly diminished DA release evoked by administration ofMETH (1 mg/kg, s. c.) compared to the wild-type mice (P<0.001 forphenotype). In the wild-type mice, the extracellular DA in nucleusaccumbens definitely increased by the administration of METH (1 mg/kg,s. c.), on the contrary, in the PTPζ-deficient mice, extracellular DAincreasing response evoked by METH stimulation attenuated obviously incomparison to that of the wild-type mice (FIG. 5A).

[0076] It was confirmed that locomotor activity after METHadministration attenuated in PTPζ-deficient mice corresponding to theextracellular DA concentration. Locomotion of freely moving animals wasanalyzed by an infrared monitoring apparatus, confirming that PTPζ^(−/−)mice exhibited a statistically attenuated METH-enhanced locomotion afterMETH stimulation compared to wild-type mice (*p<0.05). The results areshown in FIG. 5B. In other words, it was identified that the cause ofhyporesponsiveness to stimulant drugs was presented in the process ofrelease and uptake of DA from a presynaptic region. On the other hand,when DA releases from synaptic vesicles were induced by causingdepolarization by local administration of 100 mM KC1 solution throughmicrodialysis probe inserted into the nucleus accumbens, DA releaseresponse of PTPζ-deficient mice was the same level as that of wild-typemice (FIG. 5C). In addition, as there was no change in the extracellularDA concentration in a steady state as well (FIG. 5D), it was revealedthat PTPζ was not essential to the release mechanism of DA synapticvesicle.

[0077] (Role of PTPζ in DA Pathway)

[0078] It is very important to clarify the region of PTPζ expression inelucidation of the role of PTPζ in DA pathway. As detailed expressionpattern of PTPζ in mature brain is unknown, the expression region of themolecule was identified from the expression pattern of a LacZ gene, areporter gene of the PTPζ gene, in this case. In other words, cells thatwere stained blue by X-gal were identified as PTPζ-positive cells.Regional distribution of cells that display PTPζ promoter activity inthe DA circuit of adult mice was examined in adult heterozygous mice byX-gal staining (blue signal), and then by TH staining (brown signal),and the results are shown in FIGS. 6A and B. A number of positive cellswere observed in substantia nigra (SN) and ventral tegmental area (VTA),which are nuclei of origin of DA pathway,on the contrary, no cells weredetected in striatum (ST) and nucleus accumbens (NAC), which are themain regions for receiving DA projections. This result is consistentwith the result of pharmacological analysis wherein the functionalchange in the presynaptic region of DA pathway was identified bydeficiency in PTPζ.

[0079] Next, in order to confirm a possibility that PTPζ functionsdirectly in DA neurons, an analysis by RT-PCR using RNA prepared fromsingle cells as a template, that is, single-cell RT-PCR, was conducted(the above-mentioned method A-7 and A-8). Single-cell RT-PCR using cDNAderived from mouse brain as a template was conducted, then the productswere analyzed by agarose gel electrophoresis, followed by ethidiumbromide staining, and the results are shown in the top of FIG. 6C.Phosphatase domain D1 of PTPζ and PCR products of a marker gene,tyrosine hydroxylase (TH, dopamine neuron marker), glutamic aciddecarboxylase (GAD67, GABA neuron marker) and glial fibrary acidicprotein (GFAP, astroglia cell marker) were detected in each positionexpected from its mRNA base sequence, that is, PTPζ (189), TH (377),GAD67 (702), GFAP (517). The validity of these PCR products wereverified by DNA sequencing after cloning. PCR amplification from genomicDNA did not occur because primers were set to sandwich an introntherebetween. In addition, PCR amplification was not observed incontrols to which no reverse transcriptase was added. Representativeexamples being identified as TH-positive (DA neurons) and GAD-positive(GABA neurons) are shown at the bottom of FIG. 6C. As a result that 46cells were collected from substantia nigra and ventral tegmental area of4 mice and analyzed, 28 of TH-positive dopamine neurons and 6 ofGAD67-positive GABA neurons were identified. Eight cells that amplifiedneither TH nor GAD, and 4 cells wherein amplification of GFAP wasobserved were excluded from the analysis. Amplification of PCR productsof PTPζ was observed in 83% of DA cells (FIG. 6D). In other words, itwas proved that receptor-type PTPζ having protein tyrosinedephosphorylating activity was expressed in most DA neurons in brains ofadult mice.

[0080] (Change in Responsiveness to Forced Swimming Stress)

[0081] It is known that DA and 5-HT pathways are involved not only inlocomotor activity but also in emotion and stress response. In a forcedswimming stress test, mice were placed into a narrow pool underinescapable condition, and the swimming time was evaluated/analyzed intime course (the above-mentioned method A-2-3). It is presumed thatthough animals actively swim to escape from stress stimulus at first,they despair of inescapable situation and gradually become less mobile.A 15 minutes' test on the first day was conducted to induce a state ofdepression. In this test, there was no significant difference betweenthe swimming time of deficient mice and wild-type mice (FIG. 7A).However, in a test conducted 24 hours later, it was shown that thePTPζ-deficient mice swam longer than the wild-type mice (FIG. 7B).During the 15 minutes' test, mouse locomotion gradually decreased, andthe activity level during the last 10 minutes was significantly greaterin the PTPζ-deficient mice (n=8) than in the wild-type controls (n=8).That is, it was found that the PTPζ-deficient mice were hard to beacclimated to stress stimulus.

[0082] An elevated plus maze test, which is a general method forevaluating emotional behaviors, anxiety in particular, was conductednext (the aforementioned method A-2-4). In this test, mice are subjectedto ambivalent condition wherein they try to avoid the open arms of mazebecause brightness makes them anxious, while they feel like exploringthe site at the same time. In other words, animals that are susceptibleto anxiety will avoid the open arms and spend longer time in the closedarms. On the other hand, mice having a strong desire to exploration willshow more exploration behavior and in and out movement between the openand the closed arms. There was no apparent difference betweenPTPζ-deficient mice (n=9) and wild-type controls (n=12) in theirbehaviors in the elevated plus maze (FIG. 7C). Further, as to the totalnumber of entries into the arms of the maze, which is a measured valueof exploration behavior, no difference was observed between the twophenotypes as well (FIG. 7D). The two kinds of mice were evaluated ascomparable in these two factors, time spent in the open-the close arms(anxiety behavior) and the total number of entries into the arms(exploration behavior).

[0083] (Change in Exploration Behavior to a Novel Object)

[0084] Exploration behaviors to an object never seen before was examined(the above-mentioned method A-2-5). The results are shown in FIG. 8.Mobilities of PTPζ-deficient mice (PTPζ^(−/−), n=9) and wild-typecontrols (PTPζ^(+/+), n=9) in the whole area of the open field (FIG. 8A)and the central area of the open field (FIG. 8B) are presented asmean±SEM per each 3 minutes (*p<0.05, ** p<0.01). At the firstmeasurement (Trial. 1), nothing was placed in the field during the first9 minutes. It was found that both types of mice hardly stayed in thecentral area in this circumstance. Subsequently, when a block (an objectnever seen before) was placed at the center of the field, mice showedexploration behavior to the block, and remarkably increased thefrequency of going in and out of the central area. In accordance withthis increase, overall mobility also increased. Further, the graphshowed the time course decrease of exploration behaviors to the block.When comparing the two types of mice, PTPζ^(−/−) mice showed mobilitytwice or more higher than that of wild-type mice during the first 3minutes in the central area where the block was placed, and kept showingsignificantly higher mobility until the first 6 minutes passed. 24 hourslater, the second trial (Trial. 2) was conducted under the samecondition. Mobility of wild-type mice (PTPζ^(+/+)) showed almost noincrease, consequently, it was considered that the mice stoppedexploring the block, on the other hand, PTPζ^(−/−) mice showed anincrease in mobility which was considered to be caused by explorationbehavior as before. When the third trial (Trial. 3) was conducted,significant difference between the two types of mice was no longerobserved. These results indicate that PTPζ^(−/−) mice are slow inacclimating to a new circumstance.

EXAMPLE 2

[0085] (Role of PTPζ in Gastric Ulcer Formation Caused by VacA, a Toxinof Helicobacter pylori)

[0086] The role of PTPζ in gastric ulcer formation caused byHelicobacter pylori, in particular, immunohistological identification ofPTPζ in stomach, actual involvement of PTPζ as a receptor of exotoxinVacA secreted by Helicobacter pylori in gastric ulcer formation, and theusefulness of a PTPζ-deficient mouse as a negative control wereconsidered.

EXAMPLE 2-1

[0087] [Method]

[0088] (B-1 Immunohistochemical Staining of PTPζ in Mouse Stomachs)

[0089] Formalin-fixed and paraffin-embedded stomach tissue sections ofwild-type and PTPζ-deficient mice were immunostained with anti-6B4proteoglycan polyclonal antibody that recognizes the extracellularregion of PTPζ. The tissue sections were deparaffinized in xylene, anddehydrated in ethanol. The sections were washed with phosphate bufferedsaline (PBS, pH 7.6), then subjected to microwave for 15 minutes in 10mM citrate buffer (pH 6.0). The deparaffinized sections were incubatedfor 10 minutes with 0.3% hydrogen peroxide, and washed with PBS afterblocking endogenous peroxidase. Subsequently, the sections were blockedwith bovine serum albumin for 10 minutes, and washed with PBS. Thetreated tissue sections were incubated overnight with anti-6B4proteoglycan polyclonal antibody (1:2000) in Tris-HCl buffer at4.degree. C. containing a carrier protein and 15 mM sodium azide. Afterthe sections were washed with PBS, their specific antibody binding wasexamined by DAKO Envision System (DAKO Corp.) by using DAB and H₂O₂ assubstrates in accordance with manufacturer's protocol. The sections wereslightly counter stained with hematoxylin.

[0090] (B-2 RT-PCR and Western Blotting of PTPζ in Mouse Stomachs)

[0091] RT-PCR of PTPζ in mouse stomachs was conducted as follows. TRIzolReagent (Invitrogen) was used for extracting RNA from mouse stomachtissues. TrueScriptII Reverse Transcriptase kit (Sawady Technology Co.,Ltd.) and oligo dT primer (dT) 30 were used for synthesizing cDNA. PCRwas performed with 10 ng cDNA derived from RNA as a template, 10 pmol ofprimer, 0.5 U of EX-Taq polymerase (Takara Shuzo) and reaction buffersattached to EX-Taq polymerase, in a total volume of 20 μl. The PCR wasconducted under the following conditions: one cycle at 94.degree. C. for5 minutes, 35 cycles at 95.degree. C. for 30 seconds, 62.degree. C. for3 seconds, 72.degree. C. for 2 minutes, and finally one cycle at72.degree. C. for 5 minutes. In addition, the validity of the sequencesof PCR amplification products were confirmed by examining DNA sequences.PCR domains corresponding to each isoform of PTPζ are PTPζ-A type(nucleotide No. 4861-5499), PTPζ-B type (2321-2875), and PTPζ-S type(4913-5704).

[0092] Western blotting for detecting PTPζ in mouse stomachs wasconducted as follows. With Tris buffer containing 1% NP-40 and proteaseinhibitors, gastric tissues were homogenized, and after the supernatantwas obtained by centrifugation. Chondroitinase ABC treatment of thesupernatant was carried out at 37.degree. C. for 1 hour. Further,proteins were separated by SDS-PAGE (6% acrylamide gel) and transferredto PVDF membrane by semi-dry transfer. In addition, rabbit anti-6B4polyclonal antibody (1 μg/ml) and peroxidase-labeled anti-rabbitantibody were used as secondary antibodies for immunostaining to detectPTPζ. As a reaction substrate, ECL Plus reagent (Amersham Pharmacia) wasused.

[0093] (B-3 Formation of Gastric Ulcer Caused by VacA, a Toxin ofHelicobacter pylori)

[0094] After a mouse (female) of 4 weeks old was starved for 24 hours(fed water ad libitum), VacA, a toxin of Helicobacter pylori or salinewas orally administered to the mouse with a sound. 48 hours later,gastric specimens were prepared and the surface of inside wall ofstomach was observed by a stereomicroscope, and then pathologicspecimens were prepared by hematoxylen eosin staining and analyzed.

[0095] (B-4 Pathological Evaluation of Gastric Ulcer Formation Caused byVacA)

[0096] Pathological evaluation of gastric ulcer formation caused by VacAwas conducted in a following way. Epithelial damage score (EDS) wasestimated according to the following scale: 1=no lesion; 2=disarray ofcolumnar cells; 3=diffuse microerosions and epithelium disaggregation;4=erosive lesion, denudation of basal membrane, and ulceration. At EDSlevel 4, gastric mucosal ulceration was considered to have occurred. Forthe inflammation score (IS), the amounts of inflammatory cells weregraded as follows: 1=none; 2=scattered mononuclear and polymorphonuclearcells in the lamina propria and submucosa; 3=definite increase insubepithelial areas of lamina propria; 4=marked infiltration of laminapropria.

[0097] (B-5 Analyses of Phosphorylation Level of GIT1, a SubstrateMolecule of PTPζ and Cell Vacuolation in AZ-521 Cells)

[0098] In order to analyze phosphorylation level of GIT1 (Gprotein-coupled receptor kinase-interactor 1) (PNAS, 98, 6593-6589,2001), a substrate molecule of PTPζ, and cell vacuolation in AZ-521cells, uptake of neutral red and immunoprecipitation were conducted asfollows. First, AZ-521 (Health Science Research Resources Bank), acancerous cell strain derived from human stomach hypersensitive to VacA,was inoculated on a plate such that cell density was adjusted to be350,000/cm² and cultured for 24 hours, and then added withacid-activated VacA and 100 ng/ml EGF. 30 minutes after the stimulation,cell vacuolation (uptake of neutral red) and tyrosine phosphorylationlevel of GIT1 (immunoprecipitation) were analyzed.

[0099] An uptake test of the pigment, neutral red, was conducted asfollows. After components of the medium were removed, phosphate buffercontaining 0.05% neutral red and 0.5% BSA was added and made the cellsuptake it for 5 minutes. The cells were washed three times withphosphate buffer containing 0.5% BSA and then the intracellulary uptakenpigment was extracted with 70% ethanol containing 0.05% hydrochloricacid, and absorbance (540 nM) was measured.

[0100] Next, a tyrosine phosphorylation test of GIT1 was conducted asfollows. 300 μl of lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1%NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A, 1 mMsodium orthovanadate, and 1 mM NaF) was added to 1.9×10⁶ cells and theresulting mixture was left on ice for 30 minutes. After centrifuging theextract of the mixture, protein concentration of the supernatant wasadjusted to be 100 μg/ml. 25 μg sample and 25 μl Protein G Sepharose 4FF(Amersham Pharmacia) were mixed for 2 hours, and the supernatant whereincomponents nonspecifically adsorbing to gel was excluded was prepared. 2μl of rabbit anti-GIT1 antiserum was added to the supernatant, and 1hour after the reaction, 25 μl Protein G Sepharose 4FF was added andReaction was conducted for another 3 hours. After the reaction, beadswere washed three times with lysis buffer, and then specificallyadsorbing components were extracted with sample buffer of SDS-PAGE. Thissample was separated by SDS-PAGE and subsequently transferred to PVDFmembrane by semi-dry transfer, and used for immunoprecipitation.Anti-tyrosine phosphorylation mouse monoclonal antibody PY20 was usedfor the detection of tyrosine phosphorylation of GIT1 and goat anti-GIT1antibody (Santa Cruz Biotechnology, Santa Cruz) was used for thedetection of GIT1.

EXAMPLE 2-2

[0101] [Results]

[0102] (Expression of PTPζ in a Mouse Gastric Epithelial Cell Layer)

[0103] Whether PTPζ was expressed in the mouse gastric epithelial celllayer was examined by using rabbit polyclonal antibody whichspecifically recognizes the extracellular domain of PTPζ, and theresults are shown in FIG. 9 (see reference 3). As it can be seen fromthe wild-type mouse (FIG. 9A) and the PTPζ-deficient mouse (FIG. 9B), ithas been revealed for the first time that PTPζ expresses in a gastricepithelial cell layer of a wild-type mouse. The reason why PTPζ wasoriginally thought to express only in central nervous system may beexplained by the fact that the expression in stomach was too low incomparison to that in brain to be distinguished from the background. Inthe present invention, it was possible to set extremely hypersensitiveimmunostaining conditions as a result of the use of PTPζ-deficient miceas negative controls.

[0104] (Expression of PTPζ Isoforms in a Mouse Gastric Epithelial CellLayer)

[0105] The result of RT-PCR confirmed the expression of all splicevariants of PTPζ-A type, PTPζ-B type and PTPζ-S type in gastric tissue(FIG. 10A). On the other hand, Western blot analysis indicated thatreceptor-type PTPζ-B type was dominant as an actual protein component,and as eletrophoretic mobility was not changed by chondroitinase ABCtreatment, it was revealed that the PTPζ-B type expressed in the stomachis not modified with chondroitin sulfate (FIG. 10B).

[0106] (Formation of Ulcer in Mouse Gastric Epidermis)

[0107] Formation of ulcer in mouse gastric epidermis at 48 hours afterthe administration of VacA was examined with a stereomicroscope. Theresults are shown in FIG. 11. As to wild-type mice that wereadministered with VacA, 3 out of 7 mice developed apparent ulcers intheir stomachs (arrows, FIG. 11A), and in the stomachs of the other 4mice, though it was not severe, formation of erosion or apparentdisquamation of gastric epidermal layer was observed. On the contrary,in the stomachs of PTPζ-deficient mice (n=7) that were orallyadministered with VacA, no significant damage was observed (FIG. 11B).Hematoxylene eosine staining of specimens of wild-type mice administeredwith VacA showed representative ulcers (arrows in FIG. 11C with lowmagnification and in FIG. 11D with high magnification), and theappearance of the ulcer was found to closely resemble that of humanlesions caused by an infection with Helicobacter pylori. By contrast, asthe PTPζ-deficient mice were totally normal (FIG. 11E), it has beenrevealed for the first time at individual level that PTPζ plays anindispensable role in the formation of ulcer caused by VacA. As togroups to which saline was administered, both wild-type (FIG. 11F) andPTPζ-deficient mice were normal.

[0108] (Pathological Evaluation of Ulcer Formation on Mouse GastricEpidermis)

[0109] The levels of epithelial damage score (EDS) and inflammationscore (IS) were evaluated according to the method described in (B-4).Mean±SEM of EDS and IS are shown in Table 2. Epithelial damage andinflammation dependent to the dose of VacA were observed in wild-typemice (PTPζ^(+/+)), but no abnormality was observed in PTPζ-deficientmice (PTPζ^(−/−)). In the Table, the results of Mann-Whitney's U testcomparing to wild-type mice administered with saline are shown as #,P<0.05: ##, P<0.001, and to wild-type mice administered with VacA (0.5mg/kg) as **, P<0.001. Further, “nd” means that analysis was notconducted. TABLE 2 VacA n EDS IS (mg/kg) PTPζ^(+/+) PTPζ^(−/−)PTPζ^(+/+) PTPζ^(−/−) PTPζ^(+/+) PTPζ^(−/−) 0 7 7 1.1 ± 0.4 1.3 ± 0.51.3 ± 0.5 1.1 ± 0.4 0.125 5 nd 1.8 ± 0.8 nd 1.4 ± 0.9 nd 0.250 5 nd 2.2± 0.4^(#) nd 1.4 ± 0.6 nd 0.500 10  10  3.8 ± 0.4^(##) 1.3 ± 0.5^(★★)3.6 ± 0.5^(##) 1.3 ± 0.5^(★★)

[0110] (Analyses of Phosphorylation Level of GIT1, a Substrate Moleculeof PTPζ and Cell Vacuolation in AZ-521 Cells)

[0111] Tyrosine phosphorylation of GIT1, a substrate molecule of PTPζ,caused by stimuli with VacA was examined and the results are shown inFIG. 12. In VacA-hypersensitive strain AZ-521 cells, it was shown thattyrosine phosphorylation level of GIT1, a substrate molecule of PTPζdecreased dose-dependently by stimulation with VacA (FIG. 12A), and thatdecrease in phosphorylation of GIT1 was correlated with uptake ofneutral red by cells (index of vacuole formation) (FIG. 12B).Cytotoxicity of VacA was evaluated as cell vacuolation ability in vitro,and a possibility that VacA is involved in vacuole formation byincreasing the activity of PTPζ and by decreasing tyrosinephosphorylation of GIT1 has been shown.

EXAMPLE 3

[0112] (Role of PTPζ in Gastric Ulcer Formation Caused by Pleiotrophin)

[0113] Pleiotrophin is a heparin-binding secretory protein of 18 kD, andknown to be involved in neurotrophic factor-like activity, adhesion ofneurons, elongation of process and angiogenesis (BioScience JargonLibrary: “Cytokine, Growth Factor”, Yodosha, p126-127, 1998). Further,pleiotrophin is known to bind to extracellular regions of PTPζ (J. Biol.Chem. 271, 21446-21452, 1996) and alter the activity of PTPζ (PNAS, 97,2603-2608, 2000). Therefore, whether pleiotrophin is involved in theformation of gastric ulcer was examined.

EXAMPLE 3-1

[0114] [Method]

[0115] The role of PTPζ in gastric ulcer formation caused bypleiotrophin was examined as follows. After a mouse (female) of 4 weeksold was starved for 24 hours (fed water ad libitum), pleiotrophin(Peptide Institute, Inc.) was orally administered to the mouse with asound. 48 hours later, gastric specimens were prepared and the surfaceof inside wall of stomach was observed by a stereomicroscope, and thenpathologic specimens were prepared by hematoxylen eosin staining andanalyzed.

EXAMPLE 3-2

[0116] [Results]

[0117] The level of epithelial damage on mouse gastric epithelium at 48hours after the administration of pleiotrophin was examined under astereomicroscope. The results are shown in FIG. 13. Profuse bleeding wasobserved in stomachs of wild-type mice administered with pleiotrophin(FIG. 13A). By contrast, no damage was observed in stomachs ofPTPζ^(−/−) mice administered with pleiotrophin (FIG. 13B). As a resultof hematoxylen eosin staining of specimens of wild-type miceadministered with pleiotrophin, obvious damage was observed on gastricepithelium (FIG. 13C). On the other hand, PTPζ-deficient mice weretotally normal (FIG. 13D) . These results revealed for the first timethat ulcer was initiated by binding of PTPζ and a ligand molecule. Theinvolvement of pleiotrophin in gastric ulcer formation is a finding thathas never been known, and it was revealed that PTPζ-deficient mice wereextremely useful as negative controls for the formation of gastric ulcercaused by pleiotrophin.

[0118] Industrial Applicability

[0119] The present invention has shown for the first time that PTPζ isphysiologically important in central monoamine pathway, particularly, indopamine pathway. As pharmaceuticals that specifically adjust tyrosinephosphatase activity of PTPζ have not been developed yet, there is agood chance that screenings of specific pharmaceuticals with the use ofPTPζ-deficient mice will lead to the development of a novel remedy fornervous affection.

[0120] Further, the present invention has histologically shown for thefirst time that PTPζ is actually expressed in stomach, and hasdemonstrated at the level of individual mouse that PTPζ is involved inthe formation of gastric ulcer as a host receptor of VacA, a toxinsecreted by Helicobacter pylori.

[0121] It is highly possible that these results will lead to theelucidation of onset mechanism of gastric ulcer caused by an infectionwith Helicobacter pylori, and the development of novel remedies forgastric ulcer and gastritis. t,0420

1 4 1 20 DNA Artificial Sequence Description of Artificial SequenceSensePrimer 1 ggtccactga agtccacagc 20 2 22 DNA Artificial SequenceDescription of Artificial SequenceAntisense Primer 2 tctagtacaatgtatgtgcc cg 22 3 22 DNA Artificial Sequence Description of ArtificialSequencePTP-zeta Sense Primer 3 cgggagcttc ctggtcaacc ag 22 4 20 DNAArtificial Sequence Description of Artificial SequencePTP-zeta AntisensePrimer 4 agcacgggta gggagtactc 20

1. A method for screening a PTPζ inhibitor or activator wherein asubject material is administered to a non-human animal whose function ofgene DNA that encodes proteoglycan-type receptor-type protein tyrosinephosphatase (PTPζ) is deficient on its chromosome and a wild-typenon-human animal, and PTPζ activities in these non-human animals arecompared and evaluated.
 2. The method for screening a PTPζ inhibitor oractivator according to claim 1, wherein the comparison and theevaluation of PTPζ activity is the comparison and the evaluation of thefunction of central monoamine pathway.
 3. The method for screening aPTPζ inhibitor or activator according to claim 2, wherein the comparisonand the evaluation of the function of central monoamine pathway is thecomparison and the evaluation of changes in the level of centralmonoamine metabolism, sensitivity to a stimulant drug, the presence ofdysfunction of mesolimbic dopamine pathway, level of acclimation to newcircumstances, or stress-responsiveness.
 4. The method for screening aPTPζ inhibitor or activator according to claim 1, wherein the comparisonand the evaluation of PTPζ activity is the comparison and the evaluationof the level of binding to VacA, a toxin of Helicobacter pylori.
 5. Themethod for screening a PTPζ inhibitor or activator according to claim 1,wherein the comparison and the evaluation of PTPζ activity is thecomparison and the evaluation of the level of binding to pleiotrophin, aheparin-binding secretory protein.
 6. The method for screening a PTPζinhibitor or activator according to any one of claims 1 to 5, whereinthe non-human animal whose function of gene DNA that encodes PTPζ isdeficient on its chromosome is purified by being backcrossed for 4 ormore generations.
 7. The method for screening a PTPζ inhibitor oractivator according to any one of claims 1 to 6, wherein the non-humananimal is a mouse.
 8. A PTPζ inhibitor or activator obtained by themethod for screening a proteoglycan-type receptor-type protein tyrosinephosphatase (PTPζ) inhibitor or activator according to any one of claims1 to
 7. 9. The PTPζ inhibitor or activator according to claim 8, whereinthe PTPζ inhibitor or activator is a binding inhibitor between PTPζ andVacA, a toxin of Helicobacter pylori.
 10. The PTPζ inhibitor oractivator according to claim 8, wherein the PTPζ inhibitor or activatoris a binding inhibitor between PTPζ and pleiotrophin, a heparin-bindingsecretory protein.
 11. A remedy for dysfunction of central monoaminepathway containing the proteoglycan-type receptor-type protein tyrosinephosphatase (PTPζ) inhibitor or activator according to claim 8 as anactive component.
 12. A remedy for gastric ulcer or gastritis caused byHelicobacter pylori containing the proteoglycan-type receptor-typeprotein tyrosine phosphatase (PTPζ) inhibitor according to claim 8 or 9as an active component.
 13. A remedy for gastric ulcer or gastritiscaused by pleiotrophin containing the proteoglycan-type receptor-typeprotein tyrosine phosphatase (PTPζ) inhibitor according to claim 8 or 10as an active component.
 14. A non-human model animal whose function ofgene DNA that encodes proteoglycan-type receptor-type protein tyrosinephosphatase (PTPζ) is deficient on its chromosome and which ishyposensitive to a central stimulant drug (an addictive drug).
 15. Thenon-human model animal according to claim 14, wherein the non-humananimal is hyposensitive to a stimulant drug.
 16. The non-human modelanimal according to claim 14 or 15, wherein the non-human animal whosefunction of gene DNA that encodes PTPζ is deficient on its chromosome ispurified by being backcrossed for 4 or more generations.
 17. Thenon-human model animal according to any one of claims 14 to 16, whereinthe non-human animal is a mouse.
 18. A non-human model animal whosefunction of gene DNA that encodes proteoglycan-type receptor-typeprotein tyrosine phosphatase (PTPζ) is deficient on its chromosome andwhich is hyposensitive to VacA, a toxin of Helicobacter pylori.
 19. Anon-human model animal whose function of gene DNA that encodesproteoglycan-type receptor-type protein tyrosine phosphatase (PTPζ) isdeficient on its chromosome and which is hyposensitive to pleiotrophin,a heparin-binding secretory protein.
 20. The non-human model animalaccording to claim 18 or 19, wherein the non-human animal whose functionof gene DNA that encodes PTPζ is deficient on its chromosome is purifiedby being backcrossed for 4 or more generations.
 21. The non-human modelanimal according to any one of claims 18 to 20, wherein the non-humananimal is a mouse.